Surfactants in carbomycin a fermentation



United States Patent 3,483,088 SURFACTANTS IN CARBOMYCIN A FERMENTATION Donald B. Seeley, Waterford, Conn., assignor t0 Chas. Pfizer & Co., Inc., New York, N.Y., a corporation of Delaware No Drawing. Filed Jan. 16, 1967, SenNo. 609,332 Int. Cl. C12d 9/00; C12b Z/OO U.S. Cl. 195-114 6 Claims ABSTRACT OF THE DISCLOSURE Addition of soluble, non-growth inhibiting, nonionic (poly)oxyethylene glycol ether surface-active agents to carbomycin A-producing fermentation media gives rise to an increased yield of the antibiotic.

This invention relates to the production of the antibiotic called carbomycin A, and, in particular, to the use of surface-active agents in its production by fermentation.

In the process for the production of carbomycin A by fermentation, the addition to the fermentation medium of a soluble, non-growth inhibiting, nonionic (poly)oxyethylene glycol ether surface-active agent, in an amount to provide a concentration of about 0.2-4% solution of said surface-active agent in said medium, results in a substantial increase in the potency of the fermentation broth. In the instant invention, it is preferable to produce the antibiotic by cultivation of Streptomyces halstea'iz. in an aqueous, nutrient medium under submerged aerobic conditions. The instant fermentation is unlike many other antibiotic-producing fermenta'tions in that it appears that cell growth and division are required during the process in order to obtain the production of carbomycin A, thus giving rise to the requirement that the surfactant be non growth inhibiting. Also, the fermentation is unique in that the culture used has the unusual characteristic of forming tightly twisted conglomerates of growth, which resemble bulging cylinders with loose ends. The nonionic surfactants loosen these conglomerates, with the resulting potency enhancement then thought to occur because of improved transfer of nutrients at the cell surface. Some part of the effect might also be due to the increased solubility of critical intermediates.

The surfactants which are effective in the instant invention are the nonionic condensation products of a hydrophile, ethylene oxide in all present instances, and a hydrophobe, selected from straight-chain alkyl phenols and fatty alcohols, such as nonylphenol, lauryl alcohol, tridecyl alcohol, stearyl alcohol, and oleyl alcohol. By varying the weight percentage of ethylene oxide content of the surfactant, the characteristics of the surfactant can be modified to suit its use as an emulsifier, wetting agent, solubilizer, etc.; a commonly used scale for measuring the weight percentage of ethylene oxide content of the surfactant is the hydrophile-lipophile balance (HLB) number, which is equal to one fifth the weight percentage of ethylene oxide content of the instant surfactants. It appears that increasing the weight percentage of ethylene oxide content serves to decrease the degree to which the surfactant may inhibit the growth of the microorganism. Thus, a high HLB number seems to be indicative of surfactants which are non-growth inhibiting in the instant invention. Of course, depending upon the nature of the hydrophobe of the surfactant, various ranges of the HLB number prove to be effective for the various surfactants. Therefore, a minimum HLB number cannot be specified, below which all the surfactants become growth inhibiting, but generally, surfactants which have been found to be effective have HLB numbers in the range of about 14.0 to 17.5. Some surfactants with HLB numbers below this range have exhibited this same effect. Indeed, a polyoxyethylated stearyl alcohol containing tWo units of ethylene oxide per molecule has an HLB number of only 4.9, yet an enhancement of potency results from its use.

The concentration of surfactant which is effective in increasing the yield of antibiotic in the instant fermentation is surprisingly high. In other fermentation, surfactant concentrations as low as 0.001% have produced noticeable effects, and concentrations greater than about 12% have often exhibited a toxic effect and resulted in a decreased yield. However, a concentration of about 0.2-4% weight/volume of surfactant in the instant fermentation medium has been found to be beneficial. Below 0.2%, no effect can be observed, and although concentrations in excess of 4% do not appear to be deleterious, they show no advantage over concentrations within the aforesaid range. A preferred range is from about 2 to about 4%. Thus, it is necessary that a surfactant to be used in the instant invention be soluble in the fermentation medium within said concentration range. This requirement creates an upper limit on the number of ethylene oxide units per molecule of surfactant, since an increasing molecular weight serves to decrease the solubility of the surfactant.

Among the soluble, non-growth inhibiting, nonionic surfactants found to be effective are polyoxyethylated nonylphenols containing at least about 13 units of ethylene oxide per molecule (e.g., Tergitol NP-33, NP-35 and NP-4O of Union Carbide, and Hodag E-20 and E-3O of Hodag Chemical Company), polyoxyethylated lauryl alcohols containing at least about 12 units of ethylene oxide (e.g., Brij 35 of Atlas Chemical Company and Lipal 12LA of Drew Chemical Company), polyoxyethylated tridecyl alcohols containing at least about 15 units of ethylene oxide (e.g., Hodag TD-lS), polyoxyethylated stearyl alcohols containing at least about 2 units of ethylene oxide (e.g., Hodag S2 and 5-10), and polyoxyethylated oleyl alcohols containing at least about 12 units of ethylene oxide (e.g., Emulphor ON-870 of General Aniline & Film). An upper limit on said ranges of ethylene oxide units per molecule is imposed by the requirement that the surfactants be soluble within the hereinbefore specified concentration range. Preferred surfactants are polyoxyethylated nonylphenols containing about 13- 35 units of ethylene oxide and polyoxyethylated lauryl alcohols containing 12-23 units of ethylene oxide; more preferable is the use of polyoxyethylated nonylphenol containing 15 units of ethylene oxide.

Numerous surface-active agents are commercially available, as noted hereinbefore. Others can be made readily by methods generally familiar to those skilled in the art, in which a selected hydrophobe is condensed with ethylene oxide in the desired molar proportions. See, for example, Schwartz, Perry and Berch, Surface Active Agents and Detergents, vol. II, -129, Interscience, 1958, and the references cited therein.

The surfactants can be introduced into the medium directly or as an aqueous solution. Also, the entire amount of surfactant can be added initially or a fraction added initially and the remainder added during the course of the fermentation process; somewhat better results have been observed with this latter method of addition.

A further advantage unexpectedly realized by the addition of the herein specified surfactants to the fermentation medium is the resulting lack of a need to harvest the antibiotic at a critical time. Prior to the use of surfactants, it had been noted that the potency of the fermentation broth did not remain constant upon attaining its maximum Value, but rather, would begin to decrease upon reaching the peak. However, with the addition of surfactant to the medium, the fermentation broth retains its maximum or near maximum potency for a substantially greater length of time.

As noted hereinbefore, the only surfactants which are effective in the instant invention are those which are non-growth inhibiting. A convenient method for determining whether a given nonionic surfactant inhibits growth is to add the surfactant to the fermentation medium in the hereinafter described process. Approximately 24 hours after the inoculum has been added to the fermentation medium a sample of broth is taken, centrifuged (2000 rpm, min.) and the volume of the cell growth noted. If said volume of cell growth is exceeded by no more than by the volume of cell growth in a similarly treated control sample to which no surfactant has been added, the surfactant may be considered to be non-growth inhibiting with respect to the instant culture.

As already stated, use of the hereinbefore specified surface-active agents will increase the yield of antibiotic obtained from the cultivation of any carbomycin A-pro ducing microorganism. A preferred process for the production of carbomycin A (US. 2,796,379 to F. W. Tanner, Jr., et al., June 18, 1957) involves the cultivation of Streptomyces halstedii NRRL2331, preferably in an aqueous nutrient medium at a temperature of about 24-30 C., and under submerged conditions with agitation and aeration. Nutrient media which are useful for this process include a carbohydrate, such as sugars, starch, glycerol and corn starch, and source of organic nitrogen, such as casein, soybean meal, peanut meal, wheat gluten, cotton seed meal, lactalbumin, tryptone and enzymatic digest of casein. The use of enzymatic digest of casein as a nitrogen source is preferred. A source of growth substances such as distillers solu'bles, yeast extract, molasses or fermentation residues, as well as mineral salts such as sodium chloride, potassium phosphate, sodium nitrate and magnesium sulfate, and trace minerals such as copper, zinc and iron may also be utilized with desirable results. If excessive foaming is encountered during the fermentation, anti-foaming agents, such as vegetable oils, may be added to the fermentation medium. The pH of the termintation tends to remain rather constant, but, if variations are encountered, a buffering agent such as calcium carbonate may also be added to the medium.

Inoculum for the preparation of antibiotic carbomycin A by the growth of a strain of S. halstedii may be obtained by employing growth from slants of such media as Emersons agar or 'beef lactose. The growth may be used to inoculate either shaken flasks or inoculum tanks for submerged growth, or, alternatively, the inoculum tanks may be seeded from the shaken flasks. The growth of the microorganism usually reaches its maximum in about 2 to 3 days. However, variations in the equipment used, the rate of aeration, rate of stirring, etc., may effect the speed with which the maximum activity is reached. In general, from about 24 hours to 4 days is the desirable period for producing the antibiotic. Aeration of the medium in tanks for submerged growth is maintained at the rate of about one-half to two volumes of free air per volume of broth per minute. Agitation may be maintained by suitable types of agitators generally familiar to those in the fermentation industry. Aseptic conditions, of course, must be maintained throughout the preparation and transfer of the inoculum and the growth period of the microorganism.

Recovery of the antibiotic is accomplished by means generally familiar to those skilled in the art, such as extraction, precipitation and the use of strong cation exchange resins.

The present invention embraces not only the use of the hereinbefore described organism but also of mutants thereof produced by subjecting the organism to such measures as X-radiation, ultraviolet radiation, nitrogen mustard and the like.

The following examples are given to more fully illustrate the present invention. It is understood that these 4 examples are for illustrative purposes only and are not to be considered as the only manner in which the invention may be embodied.

EXAMPLE I An inoculum is prepared, using a growth medium having the following composition:

Grams Cerelose 15 Soy flour 30 Magnesium sulfate heptahydrate 1 Calcium carbonate 10 This mixture is diluted with water to one liter, pH adjusted to 6.8 with potassium hydroxide, and then sterilized and cooled. Spores of a 7-day old Emerson agar slant of S. halstedii NRRL-2331 are transferred under aseptic conditions into 20 ml. of water, and a homogeneous suspension of spores is obtained by shaking. 0.5 ml. of this suspension is transferred into 750 ml. of the above medium, and the organism grown in a shaken flask for 30 hours at 30 C.

A nutrient medium is prepared having the following composition:

Cerelose grams 25.0 Beet molasses do 15.0 Starch do 5.0 Manganese sulfate monohydrate do 1.5 Magnesium sulfate heptahydrate do 1.25 Casein digest (5%) ml 400 This mixture is diluted with water to 1 liter and the pH adjusted to 7.0 with potassium hydroxide. 0.5 m1. of DCN 1 antifoaming agent (Do-w Chemical Company) and 10 g. of polyoxyethylated lauryl alcohol containing 23 units of ethylene oxide 2 is added and the mixture is then sterilized.

ml. of the inoculum is transferred under aseptic conditions into the above nutrient medium. After agitation and aeration for two days, the potency of the broth is found to be 725 meg/ml.

Formaldehyde is added to biostabilize the broth, in an amount to provide a 0.5% v./v. solution, pH adjusted to 7.0, and the solution then extracted twice with A volume of methyl isobutyl ketone. The antibiotic is extracted from the ketone into /s volume of water at pH of about 2.0, using sulfuric acid to adjust the pH, and the aqueous phase then concentrated to /5 volume after adjusting the pH to about 4.6. The antibiotic is precipitated by adjusting the pH to about 8.3 with sodium hydroxide and separated by filtration.

EXAMPLE II Polyoxyethylated nonylphenol containing 15 units of ethylene oxide is added to the medium of Example I in place of said polyoxyethylated lauryl alcohol. The quantities of surfactant used are noted below with the corresponding broth potencies obtained therefrom.

Surfactant g./l. Potency, mcg./ ml.

'lergitol NP-35 of Union Carbide.

Dow Corning Antifoam A, a silicone-type defoamer. 2 131-1 35 of Atlas Chemical Company.

5 EXAMPLE 111 A nutrient medium having the following composition is prepared:

Cerelose grams 25.0 Beet molasses do 15.0 Starch do 5.0 Manganese sulfate monohydrate do 1.5 Magnesium sulfate heptahydrate do 1.25 Casein digest (5%) ml 400 The nutrient medium of Example I is prepared wherein said polyoxyethylated lauryl alcohol is replaced by the surfactants indicated below. After sterilization, the medium is seeded with an inoculum of S. halstedii NRRL- 2331 under aseptic conditions and subjected to agitation and aeration at 30 C. for 2 days. The broth potencies are then determined and in each case are found to be substantially greater than that of a control fermentation.

Units of ethylene oxide Polyoxyethylated nonylphenol 13 Do 20 Do 35 Polyoxyethylated tridecyl alcohol 15 Polyoxyethylated lauryl alcohol 12 Polyoxyethylated stearyl alcohol 2 DO 1O Polyoxyethylated oleyl alcohol 23 D0 23 What is claimed is:

1. In the process for the production of carbomycin A by cultivation of a carbomycin A-producing microorganism in an aqueous, nutrient medium under submerged, aerobic conditions, the improvement which comprises the addition to the fermentation medium of a soluble,

non-growth inhibiting, nonionic (poly)oxyethyleue glycol ether surface-active agent, in an amount to provide a concentration of about 2 to 4% weight/volume of surface-active agent in said medium.

2. The process of claim 1 wherein said surface-active agent is selected from the group consisting of soluble polyoxyethylated nonylphenols containing at least about 13 units of ethylene oxide, soluble polyoxyethylated lauryl alcohols containing at least about 12 units of ethylene oxide, soluble polyoxyethylated tridecyl alcohol containing at least about 15 units of ethylene oxide, soluble polyoxyethylated stearyl alcohols containing at least about 2 units of ethylene oxide, and soluble polyoxyethylated oleyl alcohol containing at least about 12 units of ethylene oxide 3. The process of claim 1 wherein said surface-active agent is polyoxyethylated nonylphenol containing about 1335 units of ethylene oxide.

4. The process of claim 1 wherein said surface-active agent is polyoxyethylated nonylphenol containing 15 units of ethylene oxide.

5. The process of claim 1 wherein said surface-active agent is polyoxyethylated lauryl alcohol containing about 12-23 units of ethylene oxide.

6. In the process for the production of carbomycin A by the cultivation of Streptomyces halstedii N'RRL-2331 in an aqueous, nutrient medium under submerged, aerobic conditions, the improvement which comprises the addition to said medium of polyoxyethylated nonylphenol containing 15 units of ethylene oxide, in an amount to provide a concentration of about 2 to 4% of said polyoxyethylated nonylphenol in said medium.

References Cited UNITED STATES PATENTS 6/1957 Tanner et al 19580 X 7/1964 Phillips 195--8O OTHER REFERENCES MAURICE W. GREENSTEIN, Primary Examiner US. Cl. X.R. 19580 

